The aim of this research program is to investigate the anti- hemostatic and anti-inflammatory compounds in the saliva of blood-feeding insects and ticks which allow efficient blood feeding and enhancement of pathogen transmission. Anti-hemostatic compounds of interest include anti-clotting, anti- platelet and vasodilators. Anti-inflammatory compounds include immunomodulatory compounds as well as compounds that modify effector arms of the immune response, such as anti-complement activity found in the saliva of some ticks. While the vector attempts to modify the feeding site to enhance success of blood feeding, such site becomes locally compromised in its ability to react to injury and becomes an easy site for pathogen invasion. On the other hand, when the vertebrate mounts an immune response to these salivary compounds, pathogen transmission may be compromised. Novel pharmaceuticals and novel targets for vaccine development will be ultimate benefits of this program. The work involves obtaining saliva or salivary glands of the insects and ticks under study, bioassay of their biological activity using both in vivo and in vitro tests, purification of the individual activities using chromatographic and electrophoretic techniques, microsequencing of the peptides and proteins, and final identification of the total primary sequence by molecular biology methods. Confirmation of the clones is found by expression in bacterial, yeast, or eukaryotic cell lines. We have initiated a reverse approach where salivary cDNA libraries from blood-feeding insects and ticks are being mass sequenced, providing new insights in the discovery of novel compounds. In the current fiscal year we continued on the discovery of gene products that are expressed in the salivary glands of 2 additional important vectors of human disease, the mosquitoes Anopheles darlingi and Culex quinquefasciatus. With this approach, we discovered novel salivary enzymatic activities, novel properties of known salivary proteins, and novel anti-coagulants with unique properties, such as the tick Penthalaris and a novel anionic lipid binding protein with prothrombinase inhibitory activity. Our bioinformatic capability also allowed us to contribute to creating a database on the proteome of the mosquito Anopheles gambiae. We have also collaborated extensively with other research groups inside and outside the NIAID. We have contributed to bioinformatic analysis of mosquito EST and microarray data from the laboratories of Dr. Marcelo Jacobs-Lorena and George Dimopoulos from the Johns Hopkins University, in genome analysis of Plasmodium falciparum and Leishmania brasiliensis with Drs. X. Su and A. Cruz, from NIAID and Ribeirao Preto, Brazil. We are also continuing the analysis of transcriptomes of P. yoelli and P. falciparum with Drs. John Sacci and Joao Aguiar, from the University of Maryland and the Naval Biomedical Institute and in trancriptome analysis of parasitic worms with Dr. Thomas Nutman (NIAID). Dr. John Andersen is also collaborating with protein purification and proteomic studies with Drs. Alan Sher (NIAID). With Dr. Fernando Cunha (State University of Sao Paulo), we are continuing to investigate pharmacological effects of peptides from tick and sand fly saliva that may act on septic shock. We are also collaborating with our expertise to Dr. Steve Hoffman's aim of producing an attenuated sporozoite vaccine to protect humans from malaria, in particular to determine salivary gland contaminants in such preparations.